Dynamic silver speciation as studied with fluorous-phase ion-selective electrodes: Effect of natural organic matter on the toxicity and speciation of silver

Highlights

• Ag⁺ binding to natural organic matter (NOM) studied time-resolved and in situ

• Quantification of Ag⁺ binding to NOM and impact of NOM on Ag⁺ toxicity

• Fast kinetics of Ag⁺ binding to NOM and strengths of binding to different NOMs

• Silver speciation affects effect of NOM on Ag⁺ toxicity

Abstract

The widespread application of silver in consumer products and the resulting contamination of natural environments with silver raise questions about the toxicity of Ag⁺ in the ecosystem. Natural organic matter, NOM, which is abundant in water supplies, soil, and sediments, can form stable complexes with Ag⁺, altering its bioavailability and toxicity. Herein, the extent and kinetics of Ag⁺ binding to NOM, matrix effects on Ag⁺ binding to NOM, and the effect of NOM on Ag⁺ toxicity to Shewanella oneidensis MR-1 (assessed by the BacLight viability assay) were quantitatively studied with fluorous-phase Ag⁺ ion-selective electrodes (ISEs). Our findings show fast kinetics of Ag⁺ and NOM binding, weak Ag⁺ binding for Suwannee River humic acid, fulvic acid, and aquatic NOM, and stronger Ag⁺ binding for Pony Lake fulvic acid and Pahokee Peat humic acid. We quantified the effects of matrix components and pH on Ag⁺ binding to NOM, showing that the extent of binding greatly depends on the environmental conditions. The effect of NOM on the toxicity of Ag⁺ does not correlate with the extent of Ag⁺ binding to NOM, and other forms of silver, such as Ag⁺ reduced by NOM, are critical for understanding the effect of NOM on Ag⁺ toxicity. This work also shows that fluorous-phase Ag⁺ ISEs are effective tools for studying Ag⁺ binding to NOM because they can be used in a time-resolved manner to monitor the activity of Ag⁺ in situ with high selectivity and without the need for extensive sample preparation.

Introduction

Silver has been estimated to be released into the environment at more than 2500 tons annually (Ratte, 1999). Since ionic silver, Ag⁺, is known to be highly toxic to bacteria, the sustainable use of silver-containing products, such as silver nanoparticles, requires a thorough understanding of the environmental toxicity of Ag⁺ ions. Silver toxicity cannot be correlated to just the total silver present. Instead, the individual silver species must be considered for a correct assessment of toxicity. One of the main mechanisms of silver speciation is Ag⁺ coordination with ligands that occur naturally in the environment. For example, Ag⁺ is known to form stable complexes with Lewis bases such as amines, halides, and thiolates. Thiosulfate, sulfide, and chloride binding to Ag⁺ have been shown to reduce the toxicity of Ag⁺ (Hogstrand et al., 1996, Janes and Playle, 1995, Leblanc et al., 1984, Rose-Janes and Playle, 2000). Consequently, the formation of silver complexes depends heavily on the environmental conditions (Ratte, 1999). For a meaningful assessment of Ag⁺ toxicity, the coordinating ligands present in any particular environment must be identified, and their effect on the Ag⁺ toxicity must be characterized.

One of the most common coordinating substances in natural soil and aquatic environments is natural organic matter (NOM; also commonly referred to as dissolved organic matter, DOM). There have been several reports of heavy metal ion binding to NOM, such as for Cu^2 +, Pb^2 +, Cd^2 +, and Zn^2 + (Di Toro et al., 2001, Hayes, 1983, Hering and Morel, 1988, Ma et al., 1998, Sikora and Stevenson, 1988). This binding results in the formation of new chemical species with altered toxicity and transport properties, which affects the bioavailability of these metals (Di Toro et al., 2001, Glover and Wood, 2005, Pagenkopf, 1983). NOM is found in environmental systems, such as surface waters, ground waters, soils, and sediments, in concentrations ranging from 1 to more than 100 mg/L (Benoit et al., 2013, Bone et al., 2012, Fabrega et al., 2009, Gatselou et al., 2014, Unrine et al., 2012). NOM results from the decomposition of plant and animal residues and is inherently a mixture of compounds without a well-defined molecular structure (Maurer-Jones et al., 2013a). NOM consists largely of humic substances (humic acids, HA, and fulvic acids, FA) but also includes non-humic substances, such as fatty acids, sterols, natural sugars, amino acids, urea, and porphyrins (Boggs et al., 1985). Humic substances have molecular weights in the range from 300 to 300,000 and have a predominantly aromatic structure. Because they have many oxygen- and nitrogen-containing functional groups, such as carboxylic, phenolic, and amino groups, they exhibit an acidic and hydrophilic character and have high metal coordinating abilities (Gatselou et al., 2014, International Humic Substances Society, 2014). Due to their abundance in the hydrosphere, biosphere, and lithosphere, and their ability to form stable complexes with metals from both natural and anthropogenic sources, humic substances are commonly used as models for studying metal and NOM speciation (Cumberland and Lead, 2009, Di Toro et al., 2001, Fabrega et al., 2009, Maurer-Jones et al., 2013a). In this study, we utilized humic substances as models for studying the effect of NOM on Ag⁺ speciation and toxicity.

Analytical methods that have been used in NOM/Ag⁺ speciation studies have been based on ion exchange equilibrium (Chen et al., 2012, Chen et al., 2013), equilibrium dialysis (Angel et al., 2013, Boggs et al., 1985, Chen et al., 2012), atomic absorption and emission spectroscopy, mass spectrometry (Benoit et al., 2013, Hou et al., 2013, Unrine et al., 2012), ion-selective potentiometry (Benoit et al., 2013, Gunsolus et al., 2015, Maurer et al., 2012, Sikora and Stevenson, 1988), and the assessment of Ag⁺ and Ag⁺-NOM complex toxicity towards organisms (Chen et al., 2013, Fabrega et al., 2009). Several reports suggest that NOM samples from various sources decrease the toxicity of Ag⁺ to various organisms (Brauner and Wood, 2002, Gao et al., 2012, Glover et al., 2005a, Glover and Wood, 2005, Kim et al., 2013, Rose-Janes and Playle, 2000, VanGenderen et al., 2003, Wirth et al., 2012). This effect is usually attributed to Ag⁺ binding to NOM, which lowers the free Ag⁺ activity and, thereby, mitigates the ability of silver to act at the sites of toxic action in organisms (Glover and Wood, 2005, Janes and Playle, 1995). On the contrary, some NOM samples were reported to have no significant effect on the toxicity of Ag⁺ to multiple organisms, and in these cases Ag⁺ binding to NOM was concluded to be insignificant (Chen et al., 2013, Fabrega et al., 2009). Surprisingly, there has been no report to date that quantitatively investigates the correlation between the extent of Ag⁺ binding to NOM and Ag⁺ toxicity. Clearly, to investigate this correlation, it is advantageous to use techniques that directly probe Ag⁺ speciation without the added complexity introduced by the choice of organism, cell culture medium, and the type of toxicity assay as is necessary in an indirect toxicology assessment.

A challenge in direct Ag⁺ speciation studies is distinguishing different silver species, i.e., Ag NPs, free Ag⁺, and Ag⁺-NOM complexes. Except for ion-selective potentiometry, all the techniques mentioned above lack this ability. To account for this lack of selectivity, specific silver species are usually isolated by several sample preparation steps, e.g., by using molecular cut-off filters (Hou et al., 2013) or centrifugation (Hou et al., 2013). Unfortunately, this sample preparation can introduce further complexity and potential errors in measurements and the interpretation of results. Such complications include silver adsorption to sample containers and interference from positively charged complexes (in the case of the ion exchange equilibrium method). Moreover, these methods cannot be used for in situ or kinetic studies due to the long analysis time resulting from the need for sample preparation (e.g., analysis times are approximately 2 h for ion exchange equilibrium methods (Chen et al., 2012) and 3 days for equilibrium dialysis (Chen et al., 2012)). Even though binding of Ag⁺ to NOM has been studied for more than a decade, and several hypotheses about its kinetics have been proposed, the kinetics of this reaction have not been investigated directly, possibly due to the lack of appropriate methodology (Glover et al., 2005a, Ma et al., 1998, Maurer et al., 2012).

Potentiometry with ion-selective electrodes, ISEs, offers selective and sensitive in situ Ag⁺ detection, requires no substantial sample preparation, is non-destructive, has fast response times, detects only non-complexed ions, and can be used for speciation and kinetics studies (Buhlmann and Chen, 2012). There have been few literature precedents with use of commercially available solid-state ISEs to study Ag⁺ speciation (Benoit et al., 2013, Maurer-Jones et al., 2013b, Maurer et al., 2012, Peretyazhko et al., 2014, Sikora and Stevenson, 1988), possibly due to the common issue of solid state ISE biofouling in biological samples (biological molecules such as proteins adsorb strongly through sulfur groups to silver halide and sulfide electrodes) (Buhlmann et al., 1998, Chang et al., 1990, Kulpmann, 1989, Park et al., 1991). ISEs with polymeric sensing membranes suffer less from adsorption, but extraction of lipophilic biological interferents into their sensing membranes can still causing biofouling of these ISEs (Frost and Meyerhoff, 2002, Ward et al., 2003). In this work, we used ionophore-doped ISEs with fluorous sensing membranes that are less susceptible to biofouling effects. Fluorous phases prepared from perfluorocarbon derivatives have low polarity and polarizability, are both hydrophobic and lipophobic (i.e., alkanes are not miscible with perfluoroalkanes), limit extraction of lipophilic interferents into the sensing membrane, and thus are less susceptible to biofouling than other polymeric membrane ISEs (Boswell et al., 2005). Moreover, fluorous-phase Ag⁺ ISEs offer exceptional Ag⁺ selectivity due to the non-coordinating and poorly solvating properties of the fluorous phase. They also exhibit fast response times (less than 1 s), making them a unique tool for environmental Ag⁺ speciation studies (Boswell and Buhlmann, 2005, Lai et al., 2010, Maurer-Jones et al., 2013b). We used these sensors to study open questions regarding the interaction of Ag⁺ and NOM, specifically the kinetics of Ag⁺ and NOM binding and the correlation between the extent of Ag⁺ binding to NOM and the resulting Ag⁺ toxicity. While the current study focuses on Ag⁺ binding to NOM, the effect of NOM on the toxicity of silver nanoparticles is also crucial for a thorough risk assessment of silver-containing products and was addressed in parallel work (Gunsolus et al., 2015).